Abstract
The constitutive and regulated secretory pathways represent the classical routes for secretion of proteins from neuroendocrine cells. Selective aggregation of secretory granule constituents in an acidic, bivalent cation-rich environment is considered to be a prerequisite for sorting to the regulated secretory pathway. The effect of selective vacuolar H+-ATPase (V-ATPase) inhibitor bafilomycin A1 on the pH gradient along the secretory pathway was used here to study the role of acidification on the trafficking of the regulated secretory protein chromogranin A (CgA) in PC12 cells. Sorting of CgA was assessed by three-dimensional deconvolution microscopy, subcellular fractionation, and secretagogue-stimulated release, examining a series of full-length or truncated domains of human CgA (CgA-(1-115), CgA-(233-439)) fused to either green fluorescent protein or to a novel form of secreted embryonic alkaline phosphatase (EAP). We show that a full-length CgA/EAP chimera is sorted to chromaffin granules for exocytosis. Inhibition of V-ATPase by bafilomycin A1 markedly reduced the secretagogue-stimulated release of CgA-EAP by perturbing sorting of the chimera (at the trans-Golgi network or immature secretory granule) rather than the late steps of exocytosis. The effect of bafilomycin A1 on CgA secretion depends on a sorting determinant located within the amino terminus (CgA-(1-115)) but not the C-terminal region of the granin. Moreover, examination of chromaffin granule abundance in PC12 cells exposed to bafilomycin A1 reveals a substantial decrease in the number of dense-core vesicles. We propose that a V-ATPase-mediated pH gradient in the secretory pathway is an important factor for the formation of dense-core granules by regulating the ability of CgA to form aggregates, a crucial step that may underlie the granulogenic function of the protein.
Highlights
The constitutive and regulated secretory pathways represent the classical routes for secretion of proteins from neuroendocrine cells
We previously reported that a chromogranin A (CgA)-green fluorescent protein (GFP) fusion protein expressed in PC12 cells is trafficked to dense-core secretory granules [22, 23], providing a tool to investigate which determinants of the secretory apparatus, for instance lumenal pH in secretory organelles, influence CgA sorting into the regulated pathway and its subsequent storage in and exocytotic release from the core of the catecholamine storage granule
Using a series of full-length or truncated domains of CgA fused to GFP or to a newly engineered form of embryonic alkaline phosphatase (EAP), the present work sought to examine the effect of selective perturbation of the vacuolar V-ATPase proton pump by the highly specific inhibitor bafilomycin A1 [24, 25] on the sorting and trafficking of the regulated secretory protein CgA in sympathoadrenal PC12 cells
Summary
We previously reported that a CgA-green fluorescent protein (GFP) fusion protein expressed in PC12 cells is trafficked to dense-core secretory granules [22, 23], providing a tool to investigate which determinants of the secretory apparatus, for instance lumenal pH in secretory organelles, influence CgA sorting into the regulated pathway and its subsequent storage in and exocytotic release from the core of the catecholamine storage granule. Using a series of full-length or truncated domains of CgA fused to GFP or to a newly engineered form of embryonic alkaline phosphatase (EAP), the present work sought to examine the effect of selective perturbation of the vacuolar V-ATPase proton pump by the highly specific inhibitor bafilomycin A1 [24, 25] on the sorting and trafficking of the regulated secretory protein CgA in sympathoadrenal PC12 cells. Our data reveal that a pH gradient over the secretory pathway is an important factor for the formation of dense-core chromaffin secretory granules in PC12 cells, perhaps by modulating the granulogenic function of CgA
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